Method for reducing impurity level in mycophenolic acid fermentation

Abstract

The present invention relates to methods for reducing impurities of mycophenolic acid during fermentation by controlling the level of carbon source during fermentation of mycophenolic acid and for the isolation and use as a standard marker of the impurity homo-mycophenolic acid.

1 . A process for reducing impurity formation during fermentation of mycophenolic acid (MPA) comprising: controlling the level of the carbon source during fermentation of MPA, wherein the carbon source is maintained at an amount of about 0.02% to about 0.8% w/w. 
     
     
         2 . The process of  claim 1 , wherein the level of carbon source is maintained at an amount of about 0.05% to about 0.5% w/w. 
     
     
         3 . The process of  claim 1 , wherein the impurities are selected from the group consisting of: MPA-IV of the following formula: 
       
         
           
           
               
               
           
         
         homo-MPA of the following formula: 
       
       
         
           
           
               
               
           
         
         and combinations thereof. 
       
     
     
         4 . The process of  claim 1 , wherein the level of the carbon source is maintained during production phase of the fermentation process. 
     
     
         5 . The process of  claim 1 , wherein the carbon source is a carbohydrate. 
     
     
         6 . The process of  claim 5 , wherein the carbon source is starch or molasses. 
     
     
         7 . The process of  claim 5 , wherein the carbon source is at least one of glucose, sucrose, maltose or glycerol. 
     
     
         8 . The process of  claim 7 , wherein the carbon source is glucose. 
     
     
         9 . The process of  claim 3 , wherein the amount of MPA-IV in the obtained fermentation broth is less than about 0.5% area by HPLC. 
     
     
         10 . The process of  claim 3 , wherein the amount of homo-MPA in the obtained fermentation broth is less than about 0.5% area by HPLC. 
     
     
         11 . The process of  claim 1 , further comprising stop controlling the carbon source prior to harvesting. 
     
     
         12 . The process of  claim 11 , wherein controlling the carbon source is stopped by stop feeding the carbon source during final 20 to 96 hours of production stage. 
     
     
         13 . The process of  claim 1 , comprising a fermentation medium containing a starting level of a carbon source (growth period) which is about 1% to about 5% w/w of the fermentation medium. 
     
     
         14 . The process of  claim 1 , comprising a fermentation broth wherein at least one of, a mineral salt, a source of nitrogen, a microbial growth factor, a source of phosphorous, or a buffer is added to the fermentation broth. 
     
     
         15 . The process of  claim 14 , wherein the mineral salt is at least one of magnesium sulfate, manganese dichloride, ferrous sulfate, zinc chloride, copper II sulfate, ammonium sulfate, potassium dihydrogen phosphate, sodium chloride or calcium carbonate. 
     
     
         16 . The process of  claim 14 , wherein the nitrogen source is an assimilable organic or inorganic nitrogen source. 
     
     
         17 . The process of  claim 14 , wherein the nitrogen source is a nitrate, urea, an ammonium salt, corn steep liquor or an amino acid. 
     
     
         18 . The process of  claim 15 , wherein the microbial growth factor is at least one of yeast extract, or vitamins. 
     
     
         19 . The process of  claim 15 , wherein the phosphorous source is potassium dihydrogen phosphate. 
     
     
         20 . The process of  claim 1 , wherein fermentation of mycophenolic acid is by a mycophenolic acid producing micro-organism selected from the group consisting of  P. brevi - compactum, P. scabrum, P. nagemi, P. roqueforti, P. patris - mei  and  P. viridicatum  or a derivative thereof. 
     
     
         21 . The process of  claim 20 , wherein the mycophenolic acid producing micro-organism is the  Penicillium  sp. strain (accession number CCM 8364) or a derivative thereof. 
     
     
         22 . The process of  claim 1 , further comprising isolating MPA from the fermentation broth. 
     
     
         23 . The process of  claim 1 , further comprising batchwise feeding of the nitrogen source. 
     
     
         24 . The process of  claim 23 , wherein the nitrogen source is glycine and/or corn steep liquor to the fermentation broth. 
     
     
         25 . A process for preparing MMF having a reduced level of impurities comprising preparing MPA according to the process of  claim 1  and converting it to MMF. 
     
     
         26 . Isolated E-8-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-isobenzofuran-5-yl)-2,6-dimethyl-oct-6-enoic acid (“homo-MPA”) of the following formula: 
       
         
           
           
               
               
           
         
       
     
     
         27 . The isolated Homo-MPA of  claim 26 , wherein it is a solid. 
     
     
         28 . The isolated Homo-MPA of  claim 27 , wherein it is crystalline. 
     
     
         29 . The isolated homo-mycophenolic acid of  claim 26  characterized by data selected from a group consisting of:  1 H NMR (400 MHz, CDCl 3 ) δ (ppm): 1.15, 1.35, 1.42, 1.63, 1.77, 1.98, 2.15, 2.43,3.39, 3.77, 5.19, 5.20, and 7.68;  13 C NMR (100 MHz, CDCl 3 ) δ (ppm): 11.5,16.0, 16.8, 22.6, 25.2,33.0,39.1, 39.4, 61.0, 70.0106.4, 116.7, 122.2, 122.5, 135.5143.9, 153.7, 163.7, 172.9, and 182.3, and combination thereof. 
     
     
         30 . The isolated homo-mycophenolic acid of  claim 26  characterized by data selected from a group consisting of: an  1 H NMR spectrum as depicted in  FIG. 6 , a  13 C NMR spectrum as depicted in  FIG. 7  and combination thereof. 
     
     
         31 . The isolated homo-MPA of  claim 26 , wherein it is a homo-MPA composition comprising MPA in an amount of less than about 5% by weight. 
     
     
         32 . The composition of  claim 31 , wherein the homo-MPA composition contains about 5% to about 0.05% by weight of MPA. 
     
     
         33 . A process of determining the presence of homo-MPA in a sample of MPA comprising carrying out HPLC or TLC of a sample of MPA with the homo-MPA as a reference marker. 
     
     
         34 . The process of  claim 33  comprising (a) measuring by HPLC or TLC the relative retention time or factor corresponding to the homo-MPA in a reference marker sample; (b) determining by HPLC or TLC the relative retention time corresponding to homo-MPA in a sample comprising homo-MPA and MPA; and (c) identifying homo-MPA in the sample by comparing the relative retention time or factor of homo-MPA as measured in step (a) to the RRT or RRF of step (b). 
     
     
         35 . A process of determining the amount of homo-MPA in a sample of MPA comprising homo-MPA and MPA by a process comprising carrying out HPLC with homo-MPA as a reference standard. 
     
     
         36 . The process of  claim 35  comprising (a) measuring by HPLC the area under a peak corresponding to the homo-MPA in a reference standard comprising a known amount of homo-MPA; (b) measuring by HPLC the area under a peak corresponding to homo-MPA in a sample comprising homo-MPA and MPA; and (c) determining the amount of homo-MPA in the sample by comparing the area of step (a) to the area of step (b). 
     
     
         37 . A method for preparing mycophenolic acid comprising: preparing a fermentation broth containing mycophenolic acid producing micro-organism; fermenting of mycophenolic acid while feeding nutrients to the fermentation broth; controlling the level of carbon source in the fermentation broth at an amount of about 0.02% to about 0.8% w/w during the fermentation stage of mycophenolic acid; harvesting and recovering the mycophenolic acid from the fermentation broth. 
     
     
         38 . An isolated compound of following structure: